Abstract The long-term objective of the proposed research is to study the role and properties of electrical synaptic transmission via gap junctions in the CNS, in particular in the auditory system. The goldfish Mauthner (M-) cell system is ideally suited for these studies since unlike mammalian electrical synapses, the experimental accessibility makes it possible to quantify in vivo changes in junctional conductance that occur under different physiological conditions and to correlate them with anatomical, ultrastructural and molecular analysis. Our progress shows that the conductance of these electrical synapses is under the fine regulatory control of glutamatergic synapses co-localized in the same terminals. Further, our progress also shows that electrical transmission is mediated by Connexin 35 (Cx35), the fish ortholog of the mammalian Connexin 36, suggesting that mammalian electrical synapses could be similarly modulated. This proposal focuses now on understanding the molecular mechanisms underlying changes in junctional conductance. We will investigate if, in analogy to the role of scaffold proteins in chemical synapses, mechanisms of exo/endocytosis involving interactions with the scaffold ZO-1 are necessary for activity-dependent potentiation of junctional conductance. Aim 1, is to investigate the association and interaction of Cx35 with the scaffold protein ZO-1. The scaffold protein ZO-1 is known to interact with many connexins to regulate their surface expression. Our preliminary results indicate that this protein co-localizes and directly interacts with Cx35 through conserved regions of both Cx35 and Cx36 carboxy-terminus. We propose to characterize direct protein-protein interactions between Cx35 and ZO-1. Aim 2, investigates the role of the Cx35/ZO-1 association in regulating electrical synaptic transmission. It is based on evidence suggesting the existence of active trafficking of gap junction channels and that potentiation could be prevented by intracellular injections of both botulinum toxin (that block exocytosis) and by peptides that interfere with Cx35/ZO-1 interactions. The proposed research addresses the novel concept that the strength of electrical synapses is dynamically modified by the activity of nearby chemical synapses. ZO-1 could become as a result of the proposed investigations the first regulatory protein identified for electrical transmission. Furthermore, its direct interaction through conserved regions of both Cx35 and Cx36 carboxy-terminus suggests that its function might underlie a fundamental and widespread property of electrical transmission, also relevant to mammalian electrical synapses. This property could be widespread and relevant to pathological conditions such as epilepsy and developmental disorders.